SHORTENED ANALOGS OF SOMATOSTATIN

ANALOGUES DE LA SOMATOSTATINE A CHAINE ECOURTEE

English Abstract

SHORTENED ANALOGS OF SOMATOSTATIN
Abstract of the Disclosure
Compounds of the formula 1 or 1a
<IMG>
(1)
<IMG>
(1a)
in which R is hydrogen or NHR1 in which R1 is lower aliphatic acyl
containing from 1 - 6 carbon atoms or benzoyl, and pharmaceutically
acceptable salts thereof are disclosed. The compounds of formulae 1
and 1a are useful for the management of diabetes and the treatment of
acromegaly in mammals. Methods for their use also are disclosed.
-1-

Claims

The embodiments of this invention in which an exclusive property
or privilege is claimed are defined as follows
1. A process for preparing a peptide of formula 1 or 1a
(1)
<IMG>
<IMG> (1a)
in which R is hydrogen or NHR1 wherein R1 is a lower aliphatic acyl having
from 1 - 6 carbon atoms or benzoyl, or a pharmaceutically acceptable acid
addition salt thereof which comprises selecting a process from the group of
processes consisting of:
(a) when a compound of formula I is required
reacting a peptide hydrazide of formula (2) Trt-SCH2CH(R)CO-Lys-
(Boc)-Asn-Phe-Phe-NHNH2 in which R is as defined herein with a reagent
which furnishes nitrous acid in situ in the presence of a strong acid to
convert said peptide hydrazide to the corresponding peptide azide and
reacting said azide with a hexapeptide of formula (3) H-Trp-Lys(Boc)-
Thr(Bu+)-Phe-Thr(Bu+)-Ser(Bu+)-NHCH2CH2S-Trt to obtain the linear
peptide of formula (4) Trt-SCH2CH(R)CO-Lys(Boc)-Asn-Phe-Phe-Trp-Lys-
(Boc)-Thr(Bu+)-Phe-Thr(Bu+)-Ser(Bu+)-NHCH2CH2S-Trt in which R is as
defined herein followed by oxidizing said linear peptide with
iodine or thiocyanogen to obtain the corresponding cyclic disulfide
derivative of formula (5)
<IMG>
<IMG> in which R is as defined herein and subsequently re-
moving all remaining protecting groups under moderately acidic conditions
to obtain the corresponding peptide of formula 1: or subjecting
37

said linear peptide (4) to treatment with either mercuric acetate, mercuric
chloride, silver acetate or silver nitrate to remove selectively the
sulfhydryl protecting groups to obtain the mercuric or disilver salt,
respectively, of the corresponding disulfhydryl derivative; converting
the latter salt to its corresponding free disulfhydryl derivative by
treatment with hydrogen sulfide, oxidizing said last-named derivative by
treatment with oxygen, 1,2-diiodoethane, sodium or potassium ferricyanide
or iodine to obtain the corresponding cyclic disulfide derivative (5) and
removing the remaining protecting groups under moderately acidic conditions
to obtain the desired peptide of formula 1;
(b) when a compound of formula 1a is required,
removing the protecting groups under moderately acidic conditions
from said linear peptide of formula 4 to obtain said corresponding peptide
of formula 1a; and
(c) when a pharmaceutically acceptable acid addition salt of said
compound of formula 1 or 1a is required,
reacting said compound of formula 1 or 1a with a pharmaceutically
acceptable acid to obtain the corresponding pharmaceutically acceptable
salt of the corresponding compound of formula 1 or 1a.
38

2. A process as claimed in Claim 1 in which the peptide hydra-
zide of formula (2) Trt-SCH2CH(R)CO-Lys(Boc)-Asn-Phe-Phe-NHNH2 in which
R is NHR1 wherein R1 is as defined in Claim 1 is prepared by acylation of
the pentapeptide of formula H-Cys(Trt)-Lys(Boc)-Asn-Phe-Phe-OMe to obtain
the corresponding pentapeptide of formula R1 Cys(Trt)-Lys(Boc)-Ans-Phe-
Phe-OMe followed by reacting said last-named compound with hydrazine hydrate
and isolating said peptide hydrazide.
3. A process as claimed in Claim 1 in which the peptide hydra-
zide of formula (2) Trt-SCH2CH(R)CO-Lys(Boc)-Asn-Phe-Phe-NHNH2 in
which R is hydrogen is prepared by reacting an activated ester of
3-tritylthiopropionic acid with the tetrapeptide of formula
H-Lys(Boc)-Asn-Phe-Phe-OMe to obtain the tetrapeptide of formula
Trt-SCH2CH2CO-Lys(Boc)-Asn-Phe-Phe-OMe, followed by reacting said
last-named compound with hydrazine hydrate and isolating said peptide
hydrazide.
4. A process as claimed in Claim 1 in which the linear
peptide (4) as defined therein is subjected to moderately acid conditions
to obtain the corresponding compound of formula 1a
HSCH2CH(R)CO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NHCH2CH2SH in which
R is as defined therein.
5. A process as claimed in Claim 1 in which the corresponding
disulfhydryl derivative as defined therein is subjected to moderately
acidic conditions to obtain the compound of formula 1a
HSCH2CH(R)CO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NHCH2CH2SH in which
R is as defined therein.
6. The process as claimed in Claim 1 wherein said linear
peptide(4) is subjected to treatment with iodine in the presence of a
lower alkanol or acetic acid to obtain the corresponding cyclic disulfide
derivative (5).
39

7. The process as claimed in Claim 1 wherein said linear peptide
(4) is subjected to treatment with iodine at from about 0° to 30°C for
about 30 to 180 minutes in a lower alkanol or acetic acid to obtain the
corresponding cyclic disulfide derivative.
8. The process according to step (a) of Claim 1 for preparing
the peptide of formula 1, as defined in Claim 1, in which R is hydrogen.
9. The process according to step (a) of Claim 1 for preparing
the peptide of formula 1, as defined in Claim 1, in which R is
NHCOCH3.
10. The process according to step (b) of Claim 1 for preparing
the peptide of formula 1a, as defined in Claim 1, in which R is hydrogen.
11. The process according to step (b) of Claim 1 for preparing
the peptide of formula 1a, as defined in Claim 1, in which R is NHCOCH3.
12. The process according to step (c) of Claim 1 wherein a
compound of formula 1 or 1a is reacted with acetic acid to obtain the
acetic cold addition salt of the corresponding peptide of formula 1 or
1a.
13. The process according to step (c) of Claim 1 wherein a
compound of formula 1 or 1a is reacted with hydrochloric acid to obtain
the hydrochloric acid addition salt of the corresponding peptide of
formula 1 or 1a.
14. A peptide of formula 1 or 1a
(1)
<IMG>
(1a)
<IMG>
in which R is hydrogen or NHR wherein R is a lower aliphatic
acyl having from 1-6 carbon atoms or benzoyl, or a pharmaceutically
acceptable acid addition salt thereof, when prepared by the process of
Claim 1, or an obvious chemical equivalent thereof.

15. A peptide of formula 1
<IMG> (1)
in which R is hydrogen, when prepared by the process of Claim 8, or
an obvious chemical equivalent thereof.
16. The peptide of formula 1
<IMG>
(1)
in which R is NHCOCH3, when prepared by the process of Claim 9,
or an obvious chemical equivalent thereof.
17. The peptide of formula 1a
HSCH2CH(R)CO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NHCH2CH2SH (1a)
in which R is hydrogen, when prepared by the process of Claim 10, or
an obvious chemical equivalent thereof.
18. The peptide of formula 1a
HSCH2CH(R)CO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NHCH2CH2SH (1a)
in which R is NHCOCH3, when prepared by the procsss of Claim 11,
or an obvious chemical equivalent thereof.
19. The acetic acid addition salt of a compound of formula
or 1a of Claim 14, when prepared by the process of Claim 12, or an
obvious chemical equivalent thereof.
20. The hydrochloric acid addition salt of a compound of
formula 1 or 1a of Claim 14, when prepared by the process of Claim 13,
or an obvious chemical equivalent thereof.
41

Description

- AHP-6491
` iO4~6;~5
Backqround of the Invention
; a. Field of Invention
- This invention relates to derivatives of the tetradecapeptide
somatostatin. More particularly, this invention concerns shortened
derivatives and salts thereof, a process for preparing saTd derivatives
and salts, intermediates used in the process and methods for using the ~ ~ -
shortened derivatives and their salts.
b. Prior Art ~;
The name "somatostatin" has been proposed for the factor
found 7n hypothalamic extracts which inhibits tne secretion of
growth hormone (somatotropin). The structure of this factor has
been elucidated by P. Brazeau et al., Science, 179, 77 (1973) and
reported to K~ve the fol10wing tetradecapeptide structure:
H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH '
,:, .
The abbreviations used herein for the various amino
acids are Ala, alanine; Asn, asparagine; Cys, cysteine; Gly,
glyc7ne; Lys, Iysine; Phe, phenylalanlne; Ser, sertne; Thr,
i threonine; and Trp, tryptophan . ¦~
The constitution of the tetradecapeptide somatostatin has
, been confirmed by synthesis; for example, see D. Sarantakis and
Z . .
W.A. McKinley, Biochem. Biophys. Res. Comm., 54 234 (1973), J. Riv7er
' et al., Compt. Rend. Ser. D, 276, 2737 (1973) and H.U. Immer et al.,
;~ Helv. Chtm. Acta, 57, 730 (1974). ¦
The important physiological activity of this tetradecapapt7de
established it as a compound of signiflcance for clinical pharmacology ¦¦~
relatlng to the treatment of acromegaly and the management of ¦
diabetes; for example, see K. Lundbaek et al., Lancet, 2, 131 (1970) and
.~
.. ~ '.
. .
. :
; '

AHP-6491
~4~625
R. Guillemin in "Chemistry and Biology of Peptides", J. Meienhofer, Ed.,
3rd American Peptide Symposium Boston 1972, Ann Arbor Science Publications,
Ann Arbor, Mich., 1972.
The linear form of somatostatin, having two sulfhydryl
groups instead of a disulfide bridge, has been prepared recently -
by J.E.F. Rivier, J. Amer. Chem. Soc., 96, 2986 tl974). He
reports that tne linear form is equipotent to somatostatin based on
the abilTty of the two compounds to inhibit the rate of secretion of -
growth hormone by rat pituitary cells in monolayer tissue cultures. -
Only recently have there been reported polypeptides, other
than tne natural hormone and its linear form having somatostatin-like ~-
activity. D. Sarantakis et al., Biochem. Biophys. Res. Comm., 55,
538 (1973) recently reported the synthesls of the somatostatin analog,
[Ala3'1A~-sornatostatin, by solid phase methods. This analog exhibited a
1 very small amount of activlty, about 0.01% of the potency of somatostatin.
P. Brazeau et al., Biochem. Biophys. Res. Comm., 60, 1202 (1974)
recently reported the synthesis of a number of acylated des-[Alal-Gly2]-
somatostat7n derivatlves by solld phase methods. ~-
¦ The present invention discloses shortened chain dertvatives
of somatostatin which show a level of activity greater than or of the same :
order as the natural hormone as well as a duratlon of activity which is
greater than that of somatostatin. Those derivatives are prepared r~adtly ~ -
by a conventent process, which includes the following advantages: the
process starts from read71y available matertals, avoids noxious reagents,
~ Ts executed facilely and utilizes easily removable protecting groups. ,
`1 The foregoing advantages and attr7butes render the peptides
of th7s 7nvention useful for the management of diabetes and for the
, -treatment of acromegaly.
, ' ~
~ 3
`1 !

~ AHP-6491
104~)625 :-
Summary of the Invention
~ The peptides of this invention are represented by formulae I
and la; formula I representing the cyclic peptides of this invention
and formula la representing the linear reduced form
~CH2CH(R~CO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NHCH2CH21 .. :-
.
(I) '
~. '
' HSCH2CH(R)CO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NHCH2CH2SH
.
(la)
in whlch R is hydrogen or NHRI Tn which Rl is lower alTphatic acyl -
; hav1ng from I - 6 carbon atoms or benzoyl. The above peptides in which
R is hydrogen are decapeptldes and those in wh;ch R is NHRI in which
Rl Is as defined above are undecapeptides.
The pharmaceutically acceptable salts of the compounds of
formulae I and la are also included within the scope of this invention.
The peptldes of this invention are prepared by a process
whlch comprises: reacting according to the azlde couplTng method a ;
flrst peptlde hydrazide of the formula ~2)
TrT-SCH2CH(R)CO-Lys~Boc)-Asn-Phe-Phe-NHNH2 (2)
l In which R is as defined above with a second peptide of the formula (3)
H-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(Bu )-NHCH2CH2S-Trt (3)
to obtaln the linear peptlde of formula (4)
Trt-SCH2CHtR)CO-Lys(Boc)-Asn-Phe-phe-Trp-Lys(Boc)-Thr(But)-phe-Thr(But)
Ser(But)-NHCH2CH25-Trt (4)
In which R is as defined herein;followed by oxidizing said linear peptide
wlth iodine or thiocyanogen to obtain the corresponding cyclic d7sulfide
derlvative of formula (5)
SlCH2CHtR)CO,-Lys(Boc~-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(Buf)-Phe-Thr(But)-Ser(But)-
; NHCH CH ~ (5) - ................................................ .
~ 2 2 ~5~
.~ .. . .
~ In which R is as defined herain and subsequently removing all
.,,~, : , :
''~ ~' ' `
:1 ` :
:~ .
~ _ 4 _
'
,, . , ... , . ,,, ~ .. ~ , .

AHP-6491
1~4~6Z5
remainlng protecting groups under moderately acidic conditions to obtain
the corresponding peptide of formula l; or followed by subjecting said
linear peptide to treatment with either mercuric acetate, mercuric chloride,
siIver acetate or silver nitrate to remove selectively -~
,, ... , . . . ., . , . .................... . ---
the sulfhydryl prolecting groups to obtain the mercuric or disiIver 1
salt, respectively, of the corresponding disulfhydryl derivative;
convertTng the latter salt to its corresponding free disulfhydryl -
derlvative by treatment with hydrogen sulfide, oxidizing said last-
named dertvative by treatment with oxygen, I,2-diiodoethane, sodium
or potassium ferricyanide or iodine to obtain the corresponding cyclic
d7sulf7de der7vative and removing the remaining protect7ng groups under
, : .
moderately acid7c conditions to obtain the desired peptide of
;, .
formula 1. Alternatively, said cyclic disulfide derivative is
reduced to said corresponding free disulfhydryl derivative by
agents known to be effective for reducing known cyclic disulfides
. :i.: . .
to their corresponding d7sulfhydryl derivatives.
A further aspect of this invent70n compr7ses the removal
of all the protecting groups from the aforement70ned 17near
peptlde or the aforement70ned d7sulfhydryl derivat7ves under moderately
ac7dtc conditions to obta7n the 17near reduced form of the pept7de
of this 7nvention of formula la,
: i . '
HSCH2CH(R)C0-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NHCH2CH25H
7n which R is as defined herein.
- . .. . .. -
The latter compound is also obtained by d7rect reduct70n
oi the cycl7c peptide of formula I by agents known to be effective -~~
for reducing known cyciic disulfides to their corresponding
~ ~ disulfhydryl derivatives. If des7red said reduced form of the cyclic
;1 pept7de 7s conver1ed to the corresponding derivative of formula I
by one of the above oxidizing agents.
'~, :-
- .
' -, ' ,
. .
,
: ':

AHP-6491
1~4~625
Details of the Invention
In general the abbreviations used herein for designating the
amino acids and the protective groups are based on recommendations
of the IUPAC-IUB Commission on Biochemical Nomenclature, see
Biochemistry, Il, 1726 - 1732 (1972). For instance, Cys, Lys, Asn,
Phe, Trp, Thr, and Ser represent the "residues" of L-cysteine, L-lysine,
L-asparagine, L-phenylalanine, L-tryptophan , L-threonine and L-serine,
respectlvely. By the residue is meant a radical derived from the
corresponding L-amino acid by eliminating the OH portion of the carboxyl;
group and the H portion of the amino group. All the amino acids have
the natural L-configuration.
A number of procedures or techniques for the preparation of -~
peptides have hitherto been well established. For instance,the
~ ..
functional groups which are not involved in the peptide
bond formation reaction are optionally protected by a protecting
group or groups prior to the condensation reaction. For example,
protectlng groups which may be chosen for an amlno function of a
peptlde or amlno acid not involvéd In the peptide bond formation
are: the alkoxycarbonyls which include benzyloxycarbonyl (represented
by Z), t-butoxycarbonyl (represented by Boc), a,~-dimethyl-3,5-
dimethoxybenzyloxycarbonyl (represented by Ddz), 2-(D-biphenyl)-
isopropyloxycarbonyl (represented by Bpoc), D-chlorobenzyloxy-
carbonyi, -methoxybenzyloxycarbonyl, isopropyioxycarbonyl, or ethoxy-
carbonyl-; the acyl type protecting groups which include formyl, tri-
fluoroacetyl, phthalyl, acetyl (Ac), or toluenesulfonyl; the alkyl
1;- . : -
~', ;' '-''
.
.. ~ ~ ~ '
.'' . i ~- .
:.
~ ~ -6-
- . -:: , . . - . . . - . ~ . .
.. , . . .. , ~ .

AHP-6491
. 1~4~625
type protecting groups whîch include triphenylmethyl or trityl (repres- -
ented ~y Trt) or benzyl; the preferre~ protecting groups and in the process
of this invention are benzyloxycarbonyl,t-butoxycarbonyl, triphenylmethyl
and ~,~-dimethyl-3,5-dimethoxy-benzyloxycarbonyl. The protecting groups
for the hydroxyl of serine and tyrosine are represented by acetyl-, tosyl,
benzoyl, tert-butyl (represented by Bu ), trityl, and benzyl; the ~ ;
preferred protecting group ~is tert-butyl. The protecting grou~ on~
the sulfur of cysteine or modified cysteine are illustrated by benzyl,
triphenylmethyl or trityl (represented by Trt), benzyloxycarbonyl, or ~ -
acetamidomethyl (represented by Acm); the preferred protecting ~-~
groups are trityl and acetamidomethyl. The carboxylic acid
function of a peptide or amino acid can be considered protected by
a lower alkyl or lower aralkyl ester which include methyl ~
... . ..
(r~presented by OMe), ethyl (represented by OEt),or benzyl (represented
by OBzl); and also by substltuted hydrazides which include
t-butoxycarbonyi hydrazide (represented by NHNH Boc~, benzyloxycarbonyl
hydrazide (represented by NHNH Z), or ~ dlmethyl-3,5-dlmetnoxybenzyloxy-
carbonyl hydrazlde (represented by NHNH Ddz).
To promote facile condensation of the peptide carboxy
group with a free amino group of another peptide to form a new
. . ~
,1~ peptide bond the terminal carboxyl group must be activated. ¦
Descriptions of such carboxyl-activating groups are found in general
,~ textbooks of peptide chemistry; for example K.D. Kopple, "Peptides
and Amino Acids", W.A. 8enJamin, Inc., New York, 1966, pp. 45 - 51
l;~ and E. Schr~der and K. LUbke, "The Peptides"; Vol. 1, Academic Press,
~ New York, 1965, pp. 77 - 128. Examples of the activated form of the
,t,~ terminal carboxyl are acid chloride, anhydride, azide, activated ester,or
o-acyl urea of a dialkylcarbodiimide . The following activated
', ' '
.'
.
~ _7_
: -- -. - . . :; - .
,
,: : - . ' . .' . , . . . : , .
. :
: - , -. ~ ' ' ' ': : ` .
', ,

AHP-6491
62S
esters have proved to be particularly suitable in the process of
this invention: 2,4,5-trichlorophenyl (represented by OTcp), penta-
chlorophenyl (represented by OPcp), p-nitrophenyl (represented by
ONp), or t-benzotriazolyl; the succinimido group is also useful for
such acttvat;on.
The term "azide method" as used herein refers to the method
of coupling two peptide fragments which comprises the reaction of a
peptlde hydrazide with a reagenT which furnishes nitrous acld in
sltu. Suitable reagents for thts purpose include organic nitrites
(e.g. t-butyl nitrite, isoamyl nitrite) or alkali metal nitrite salts
~e.g. sodium nitrite, potassium nitrite) in the presence of a strong acid
such as hydrogen chlor7de or su7furic or phosphoric acid. The corresponding
peptide azide thus obtained is then reacted wtth a peptide having a
free amino group to obtain the desired peptide. Preferred conditions ,
for the azide method of coupling comprTses react7ng the peptide
hydrazlde with nitrous acid, generated in situ from an organic nitrite ;
!n the presence of a strong acid, preferab~y hydrogen chloride,
tpH ranglng usually from 0.1 to 2), In an anhydrous Inert organic
solvent, for example, dlmethylformamlde, dimethyl sulfoxide, ethyl
acetate, methylene dichloride, tetrahydrofuran , dioxane, and the
like at -30to 20C, preferably at about -15~ for 10 to 30 minutes
to obtain the corresponding azide. The peptide azide can be isolated
and crystallized and is preferably allowed to remain in the react70n mlxture.
Thereafter the azide in the above mixture is reacted with the peptide
.i ~ .
unit having the free amino group at temperatures ranging from -30C to
20C for about one to two hours and then at 0to 30C for 10 ta 30 hours.
An acid acceptor, preferably an organic base, for example N-ethyldi7so- ~
propylamine, N-ethylmorpholine or tr7ethylamine, is present in the reaction -
mixture in order tP make the reaction medium slightly alkaline, pre-
, ferably pH 7.0 to 7.5. See also the above cited textbooks of Kopple
.
.'~ ' ' . .
- 8 -
.. . . .
~' ': ', " , . . : '
.- . . . . : .

:
AHP-6491
1C~4~6ZS
or Schr~der and LUbke for additional descriptions of this method.
The terms "peptide, polypeptide, tripeptide, hexapeptide,
and the like" as used herein are not limited to refer to the respective
parent peptides but are al50 used with reference to mod7fied peptides
having functionalized or protecting groups. The term "peptide" as
used herein is used with reference to a peptide with tw~ to eleven
amino acid residues. In addition the res7due "SCH2CH(R)C0" as used herein
is used in reference to the restdue of an acylated or benzoylated cysteine
when R is NHRI in which Rl is as defined above or to a modified residue
of cysteine when R is H.
., . , . ':.. ,:'
The abbreviation Me represents a methyl group and NHNH2
represents a hydrazide group.
The term "lower alkyl" as used herein contemplates hydro-
, , ,.,
carbon radicals having one to three carbon atoms and includes ~ethyl,
ethyl and propyl.
The term lower "aliphatic acyl having from I - 6 carbon atomsl'
reprèsent straight or branched chain acyl groups and includes formyl,
acetyl, proplonyl, butyryl, Isobutyryl, pivaloyl, n-hexanoyl and
the like.
- :' ,. .
,~ ~ The term "mineral acid" as used herein contemplates the
strong inorganic acids and includes hydrochloric, hydrobromic,
sulfuricj or phosphoric acid. When the term is used in con-
Junction with an anhydrous system, anhydrous hydrogen chloride is the -
preferred ~ineral acid. ~;
'~ The term "miIdlyacidic conditions" as used herein con-
,~ templates conditions in which a dilute aqueous solution of an organic acid,
for oxample 30 - 80% aqueous formic, acetic or propionic acid, pre-
ferably 70 - 80%, or mixtures thereof, is a principal component of :
the reaction medium.
'. . :
.. . . . .
~ g
, . .

AHP-6491
1~4~6ZS ~:
The term "moderately acidic conditions" as used herein
contemplates conditions in which concentrated organic acids or
solutions of the mineral acids are used as a principal component
of the reaction medium at temperatures ranging from abouf -30 to
30C. Examples of preferred conditions in this case include the
use of 50 to 100% trifluoroacetic acid at 0 to 30C or 0.1 - 12N
hydrochlorlc acid in aqueous solution or in solution in an organic
solvent, or hydrogen chloride in solution in anhydrous organic solvents
at -20 to 10C.
The term "organic nitrite" includes the commercially
available alkyl nitrites, for instance, t-butyl nitrite, isoamyl
nitrite, and the like. -
The term "organic base" as used herein includes triethyl-
amine, N-ethylmorpholine, N-ethyldiisopropylamine and the like.
The term "strong base" as used herein contemplates both
organic bases as described above and strong inorganic bases
~ 7nc!uding the hydroxides and carbonates of sodium and potassium.
i The peptides of this inventlon, 7ncluding the cyclic and the
Itnear reduced forms thereof, are obtained 7n the form of the free base
or an acid addition salt either directly from the process of this
invention or by reacting the peptide with one or more equivalents of
,, .
the appropriate acid. Examples of preferred salts are those with
pharmaceutically acceptable organic acids, e.g. acetic, lactic,
succtnic, benzoic, salicylic, methanesulfonic or toluenesulfonic
acid; as well as polymeric acids such as tannic acid or carboxymethyl
cellulose, and salts with inorganic acids such as hydrohalic acids, ¦
e.g. hydrochloric acid, or sulfuric acid, or phosphoric acid . It ¦
should be noted that the peptides have two basic nitrogens giving I
,. . ~ .
rise to addition salts with one to possibly two equivalents of acid.
If desired,a~particular acid addltion salt is converted into ¦
another acid addition salt, e.g " a salt with a non-toxic,
pharmaceutically accsptable acid, by treatment with the appropriate
!1
~1.
-- ¦ O -- ! ~
:' ~: ''' ' . . . . ..

AHP-6491
104~625
ion exchange resin in the manner described by R.A. Boissonas
et al., Helv. Chim. Acta, 43, 1349 (1960). Suitable ion exchange
resins are cellulose based cation exchangers, for example carboxy-
methylcellulose or chemically modified, cross-linked dextran cation
exchangers, for example, those of the Sephade~ C type, and strongly
basic anion exchange resins, for example those listed in J.P.
Greenstetn and M. Winitz Chemistry of the Amino Acids , John
Wiley and Sons, Inc., New York and London, 1961, Vol. 2, p. 1456.
The peptides produced by the process of this Invention,
as well as thelr corresponding pharmaceutically acceptable salts, --
are useful because thsy possess the pharmacological activity of
the natural tetradecapeptide somatostatin. Their activity is
, demonstrated readily in pharmacological tests such as a modification
~A.V. Schally et al., Biochem. Biophys. Res. Commun., 52, 1314
(1973); J. Rivier et al., C.R. Acad. Sci. Paris, Ser. D, 276, 2737
(1973)~ of the in vitro method of M. Saffran and A.V. Schally, Can.
J. Biochem. Physiol., 33, 405 ~1955).
The activlty of the peptldes of formula I or la of thls
Inventlon Is demonstrated also Tn vivo in a modification of the
pentobarbital-induced increase in plasma growth hormone level in - -
the-rat as describes by Brazeau et al., cited above. In this
~t~ test the peptides of this invention show a level of activity which
is greater than or of the same order as somatostatin.
The peptides of this invention are useful for the treat-
ment of acromegaly and related hypersecretory endocrine states and in the
management of diabetes in mammals; 5ee for example, P. Brazeau et al.,
cited above. When the peptides or salts thereof are employed for such
treatment or management, they are administered systemically, preferably
* Sephadex is a trade mark
,1 .
:,j :
I ~' ' '' ~ -Il- ' ' .
:-. ~ . . .. . . ~ . ;. . : . , :
.. : - . . .. . . .. , . ~ . .. : , .

~ AHP-6491
11~)4~625
parenterally, in combination with a pharmaceutically acceptable tiquid
carrier. The peptides of formula 1 or ia have a low order .
of toxicity. The proportion of the peptide or salt thereof is
determined by its solubility in the given carrier, by the given
carrier, or by the chosen route of administration. When the peptide
or a salt thereof is used in a sterile aqueous solution, such solution may
also contain other solutes such as buffers or preservatives, as well
as sufficient pharmaceutically acceptable salts or glucose to make
the solution Tsotonic. The dosage will vary with the form of adminis-
tration and with the particular species to be treated and is preferably .
kept at a level of f~om I mcg to 303 mcg per kilogram body weight.
~owever, a dosage level in the range of from about I mcg to about 50 mcg
per kilogram body wetght is most desirably emp!oyed in order to achieve
effective results. ' !
....
The peptides'or salts thereof may also be administered in ~ '
one of the long-act1ng, slow-release or depot dosage forms described
below, preferably by intramuscular inJection or by imp1antation. Such
dosage forms are designed to release from about 0.1 mcg to about 50 mcg ''''
per kl'logram body weight per day.
It is often desirable to administer the agent continuously
over prolonged periods of time in long-acting, slow-release or depot
dosage forms. Such dosage forms may either contain a pharmaceutically ¦
acceptable salt of the peptide having a low degree of solubility in ¦
body fluids, for example one of those salts described below, or they
may contain the peptide in the form of a water-soluble salt together
with a protective carrier which prevents rapid release. In the latter
case, for example, the peptide may be formulated with a non-antiaenic
partially nydroly~ed gelatin in the form of a viscous liquid; or the
peptide may be abosrbed on a pharmaceutically acceptabieJsolid carrier,,
for example, z7nc hydroxide, and may be administered in suspension in a
pharmaceutically acceptable liquid vehicle; or the peptide may be '
formulated in gels or suspensions with a protective non-antigenk
- 12 -

AHP-6491
1~4~625
hydrocollotd, for example sodium carboxymethylcellulose, poly-
vinylpyrrolidone, sodium alginate, gelatine, polygalacturonic acids,
for example, pectin, or certain mucopolysaccharides, together wlth
aqueous or non-aqueous pharmaceutically acceptable liquid vehicles,
preservatives, or surfactants. Examples of such formulations are
found in standard pharmaceutical texts, e.g. in Remington s
Pharmaceutical Sciences, 14th Ed., Mack Publishing Co., Easton;
; Pennsylvania, 1970. Long-acting, slow-retease preparation of the ~;
peptlde produced according to the process of this invention may a!so
be obtained by mlcroencapsulation in a pharmaceutically acceptable
coating, for example gelatine, polyvinyl alcohol or ethyl cellulose.
Further examples of coating materials and of the processes used for
m7croencapsulation are described by J.A. Herbig in Encyclopedia of
Chemical Technology , Vol. 13, 2nd Ed., Wiley, New York 1967, : ~ -
pp. 436-456. Such formulations, as well as suspensions of salts of
the peptlde which are only sparingly soluble in body flu7ds, for ; ~-
example salts with pamoic acid or tannic acid, are designed to :
release from about 1.0 mcg to about 100 mcg of the active compound
per kllogram body welght per day, and preferably adminlstered by
Intramuscular tnJectlon. Alternatlvely, some of the solld dosage
forms listsd above, for example certain sparingly water-soluble
salts or dispersions in or adsorbates on sol7d carriers of salts 1
~; ~ of the peptide, for example dispersions in a neutral hydrogel of a
polymer of ethylene glycol methacrylale or similar monomsrs cross-
llnked as described in U.S. Patent 3,551,556 may also be formulated
. . .
in the form of pellets releasing about the same amounts as shown
above and may be implanted subcutaneously or intramuscularly.
Process
The process of thls invention will be illustrated by the
fo!l ow ing embodiments 7n which specific peptides of formulae I and la
are prepared.
: -
~ .
'' . '
.. ~
13 1

AHP-6491
10406ZS
ta) Compounds I and la (R = NHRI in which Rl is as defined above)
The requisite first peptide hydrazide of formula (2) ;
Trt-SCH2CH(R)CO-Lys(80c)-Asn-Phe-Phe-NHNH2 in which R is NHR in
which Rl is as defined above or alternatively written with -~
formula (2a) as Rl-Cys(Trt)-Lys(~oc)-Asn-Phe-Phe-NHNH2 is prepared
by acylatTon of the pentapeptide of formula H-Cys(Trt)-Lys(Boc)-
Asn-Phe-Phe-OMe to obtatn the pentapeptide of formula Rl-Cys(Trt)-
Lys(80c)-Asn-Phe-Phe-OMe which is subjected to hydrazinolysis to
- ';: '
obtaln said first pentapeptide hyarazlde. - ;
In a preferred embodiment of the preparation of the above
first pentapeptide hydrazide (2), a mixture of substantially
.: :
equTmolar amounts of an organic base, preferably N-ethylmorpholine,
and the pentapeptide of formula H-Cys(Trt)-Lys(80c)-Asn-Phe-Phe-OMe,
prepared as described by H.U. Immer et al., cited above, in an
Inert organic solvent, preferably dimethylformamide or tetrahydro-
furan,` at about 0 to 10C, Is treated with an excess, preferably
1.1 to 2 molar equlvalents, of the desired p-nltrophenyl acylate
or benzoate, e.g. p-nltrophenyl acetate, prepared as described by
F.D. Chattaway, J. Chem. Soc., 2495 (1931). The mixture is kept at
' I - .
0 fo 10C for about 15 to 30 hours and evaporated. The residue is ;
taken up 7n a polar organ7c solvent, preferably methanol, and -
.,~. . . .
' slowly added to a non-polar organic solvent, preferably diethyl -
ether. The residue is collected and crystallized to obtain the
t
'~,'7~ corresponding acylated or benzolated pentapeptide, e.g. the penta-
peptlde of formula Rl-Cys(Trt)-Lye(80c)-Asn-Phe-Phe-OMe in which
Rl is as defined above. Said last-named compound is dissolved in an
~ ~ inert organic solvent, for example methanol, ethanol, dimethy!formamide,
.; ' ,~ ,: '' .' : ' -
., . i~.
~~~ 14 1~ ~

AHP-649 1
1!~)41~625
and the like, preferably methanol. The solution is treated with ' ''
an excess of hydrazine hydrate, for example 15 to 30 molar
equivalents. The reaction mixture is kept at about 0 to 10C ~ ~ ;
for about 40 to 60 hours. The precipitate is collected and dried
to yteld said first pentapeptTde hydrazide of formula (2) or (2a) '
In wh7ch Rl Is as defined above, for example the pentapeptide ~
hydrazide of formula ~2) or (2a) in which Rl is acetyl. ~'
In the next step of the process of this inventlon the afore-
mentioned first pentapeptide hydrazide (2) and a second vtz., the ;
hexapeptide of formula (3) H-Trp-lys(i3Oc)-Thr(But)-Phe-Thr(Bu )- '
. . .-.
Ser(i3ut)-NHCH2CH2STrt ~descrtbed in U'.5.'Patent
No. 3,917,581, issued November 4, 1975) are coupled '
according to the azide coupling method to obtaln the corresponding
linear undecapeptTde of formula ~ in which R is NHRI as defined
above alternatively written wlth formula (4a) as Rl-Cys(Trt)-Lys- '
ti30c)-Asn-Phe-Phe-Trp-~ys(Boc)-Thr(8ut)-Phe-Thr(But~Ser(13ut)- . .'
NHCH2~i2S-Trt,for exampie the llnear undecapeptlde of the above
formula ~4a) In whlch R Is acetyl.
. j .
A convenlent and efficaclous procedure for this STap
comprlses dissolving the first pentapeptide hydrazlde (2) in
whTch R Is NHR whereln Rl Is as'deflned above 1~ an organic
solvent, preferably dimethylformamide and cooling the mlxture to
about -20 to -10C. A solution of about two to flve molar
equlvalents of a strong acid in an inert organTc solvent,
preferably three molar equlvalents of hydrogen chloride in ethyl
acetate, is added to the above solution, followed by 1.0 to 1.5 .
molar equivalents of an organic nttrito, for example, 1.2 molar ~ '
., ~,,, ,, . i'
..... .

AHP-6491
1~4~6Z5 ~ -
equivalents of t-butyl nitrite. In thTs manner the corresponding
pentapeptide aztde of formula R -Cys~Trt)-Lys(Boc)-Asn-phe-phe- _
N3 in which Rl is as defEned above, for example, acetyl, is obtained.
After about 10 to 20 minutes at about -20 to 0C, a solution of
substantially one molar equivalent of the above second hexapeptide
- and an organic base in an tnert organlc solvent, preferably two to
four molar equTvalents of N-ethyldl1sopropylamine in d7methyl-
formamide, cooled to about -20 to 0C, is added to the above
solution contaTnTng said azide. The reaction mixture is then stirred
at about -20 to 0C for one to two hours and then at about 20 to
30C for 20 to 30 hours. The solvent is evaporated under reduced
pressure. The resTdue is trTturated wlth cold dilute aqueous citric
¦ acid, water, and methanol, and separatlon of the soltd g1ves the
aforementioned linear undecapeptide of formula (4) In which R Ts
NHRI as defined above, alternatiuely written as formula (~a) in
whlch Rl Is as deflned above.
The aforementioned requlslte second hexapeptlde descrlbed
in U.S. Patent 3,917,58J, cited above, ls obtained-readlly
by coupllng accordlng to the azlde coupllng method the hexi~peptlde -
hydrazide of formula Ddz-Trp-Lys(Boc)-Thr(But)-Phe-Thr(8ut~er(8ut)-
NHNH2, prepared as described by H U. Immer et al., cited above, with
2-tr1tylthioethylamlne to give the hexapeptide of formula Ddz-Trp-Lys-
(Boc~-Thr( 8ut)-Phe-Thr(But)~er(But~NHCH2CH2STrt. Treatment of the
Iatter compound under miIdly acldlc condltions affords said second
hexapeptide of formula (3).
~ The conversion of the precedlng llnea~ undecapeptlde of
i~ ; formula (4) or (9~ obtained as described above In whlch Rl is
- -
:~ :
as def;ned above, for example acetyl, to the corresponding compound
~ . of formula I (R = NHRI) is accompll~ihed convenlently and
'i'! ~ efficiently by first subjecting the linear undecapeptlde to the

AHP-6491
. ~',''
.
1~)4~6ZS
action of iodine, preferably in the presence of a lower alkanol
or acetic acid, whereby removal of the sulfhydryl protecting
groups, i.e. Trt, and concomitant formation of the disulfide `
bridge occurs to give the corresponding cyclic disulfide
derivative of formula t5) in which R is NHRI, alternatively
written with formula (5a) as Rl-Cy~s-Lys(~oc)-Asn-Phe-Phe-Trp-Lys-
(Boc)-Thr(But)-Phe-Thr~But)-Ser-t8ut)-NHCH2CH2~ in which Rl is
i as defined above, for example acetyl. Subsequent treatment of
~he latter compound under moderately acidic conditions removes
the remaining protecting groups (i.e. Boc and But) to give the
corresponding cyclic peptide of formula I in which R is NHR
~ 7n which R is as described above, for example acetyl.
j In a preferred embodiment of the above transformation
~I the llnear undecapeptTde of formula (4) is dissolved in acetic
acid or methanol, ethanol or other suitable lower alkanol,
for example, propaool, isopropanol or butanol. This solution
19 added to an excess of iodine ~5 to 25, preferably 10 malar
equivalents) dissolved in a lower alkanol, preferably 2 - 5% iodine
in methanolj. The t7me and temperature of this reaction is not
,., ...
crltlcal; however, it is deslrable to keep the reaction between
0 and 30C by regulating the addition to the iodine solution or
by cooling of the reaction mixture, or by a combination of both.
~- .
:`lL -~ : Under these conditions the addition usually~takes 30 to 60 minutes. I
~ J7r~
After the addition of iodine the mixture is stirred at 20~to 50C
tor 30 to 120 minutes, preferably for 60 minutes. Thereafter the
; mlxture is cooled to about 0C and an excess of a miId reducing
agent, preferably sodium thiosulfate in aqueous solution, is added
i , .
- ~
. ~ ,.
. '
- 17 -
-, . . . . . .

AHP-6491
'
16)4~625
in order to destroy excess iodine. The mixture is concentrated
and the residue is suspended in water. Collection of the solid
material affords the desired corresponding cyclic disulfide
derivative of formula (5) in which R is NHRi and Rl is as
defined above, for example the compound of formula (5) in which
R is NHCOCH3 in which the Boc and But protecting groups are still
present.
Alternatively, the linear undecapeptide (4) is converted
to the aforementioned corresponding cyclic disulfide (5~
der7vatTve by the method of R. G. Hiskey and R. L. Smith, J. Amer.
Chem. Soc., 90, 2677 (1968) using thiocyanogen.
Again alternatively, the above cyclic disulfide derivative
(5) ls also obtained by selectively removing the sulfhydryl
1 protectlng groups of the above linear undecapeptide (4) by the
,d actlon of a mercuric or silver salt, for example, mercuric acetafe,
mercurlc chlor7de, silver acetate or stlver nltrate, in an inert
organlc solvent, for example dlmethylformamlde or acetlc acid, ~ -
accordtng to known methods; for example, see B. Kamber, and
W. Rittel, Helv. Chem. Soc. 87, 4922 ~1965) and R. G. Denkewaiter -
et al., J. Amer. Chem. Soc., 91,-502 (1969). The corresponding
mercurtc or disiIver salt is then converted by hydrogen sulfide -~;
treatment to the corresponding free disulfhydryl derivative, see
L. Zervas et al., cited above. The latter derivative is then
converted to the aforementioned cyclic dTsulfide derivative (5)
by treatment with a mild oxidizing agent, for example i odI ne
accordlng to the method descrihe~i hereinbefore, or oxygen according
- 18 -
... .

AHP-6491
. '; ~ '
104~1625
to the method of J. Rivier et al., C. R. Acad. Sci. Ser. D, 276,
2737 (1973), or 1,2-diiodoethane according to the method of
F. Weygand and G. Zumach, Z. Naturforsch. 17b, 807 (1962), or
sodium or potassium ferricyanide according to the method of -- -
D. Jarvis et al., J. Amer. Chem; Soc., 83, 4780 (1961).
Finally, the aforementioned cyclTc disulfide derivative
of formula (5) in which R is NHRI and Rl is as defined abo~e, is
transformed into the cyclic undecapeptide of formula I (R = NHRI
and Rl Is as defined above) by subjecting the former to moderately
acidTc conditions whereby the remaining protecting groups of the
cycllc disulfide derivative are removed. Generally this step is
carried out by dissolving the cyclic disulfTde derivative in an ,-
:,
aqueous reaction medium containiny a strong acid at 0 to 20C
for 10 to about 60 minutes. Examples of such medTa are 80 to 100%
'1 trifluoroacetic acid, 10 to 20~ aqueous sulfuric acid, 10%
`1 phosphoric acid, 10 - 30S hydrobromtc acid or 10 to 36% hydro-
chloric actd. An extremely useful medlum is concentrated -
hydrochlorlc acid. Preferred conditions for this step include 1 -
dlssolving the cyclic disulfide in a minimum of concentrated
hydrochloric acid çooled to 0C and stirring the mixture at 0C
for five to ten minutes under a nltrogen atmosphere. Thereafter
glacial acetic acid (10 vols.) is added, the solution is cooled
to about -70C and Iyophilized to give the cyclic undecapeptide
~; of formula I (R = NHRI and Rl is as defined above), for example
~A~; . the compound of formula I in which R is NHCOCH3. The latter
product is purified further by ion exchange chromatography, , ~ -
~;~ preferably using a carboxymethyl cellulose cation exchanger and
~ .
'~
`' ' - 1 9 _

-~ AHP-6491
1~14~1625
i:
ammonium acetate as the eluant. In this case the peptide is
obtained in the form of its acid addition salt with acetic
acid. Alternatively, the peptide ts purified by partition ~ j
chromatography on a chemically modified cross-linked dextran;
for example, Sephadex*LH-20 or Sephadex*G-25. In the case where
Sephadex*LH-20 is employed and methanol as the eluting solvent,
the peptide is obtalned Tn the form of Its acid addltion salt
wlth hydrochlorlc acid. In the case where Sephadex G-25 and
acetlc acld or acetic acld-water-butanol is employed, the peptide
~i Is obtained in the form of its acetic acid addltion salt. The
1 latter salt, when subJected to repeated IyophTllzation from water ~ -
~ ylelds the cycllc undecapeptlde of formula I (R z NHRI and Rl is
I as deflned above), for example the cyclic disulfide of acetylcysteinyl- -~
' Iysylasparaginylphenylalanylphenylalanyltryptophyllysylthreonylphenyl-
alanylthreonylseryl-2-thioethylamlde, in the form of the free base.
The linear reduced form of the cycllc undecapeptlde of formula I
R = NHR and Rl Is as deflned above) is obtalned preferentially by
removal of the protectlng groups from the aforementloned llnear
undecapeptlde of formula ~4~ In whlch R Is NHR~ and Rl i5 as defined .
above. Convenient conditlons fo~thts deprotectlon srep comprlse ~-
dissolvlng the linear undecapept!de ~4) Ih concentrated hydro-
chloric acld at about 0 to 5C In sn Inert atmosphere, for ~ -
example, nitrogen or argon. The mlxture Is kept at this tempera-
3 ture for five to~10 minutes. Subsequent isolation of the ITnear
; reduced form (la, R = NHRI and Rl is as defIned above) is
~; accomplIshed in the same manner as described prevlously for the
; Isolation of the cyclic undecapeptide (I; R = NHRI and Rl is as
defined above).
* Sephadex is a trade mark
- ~ . . ~ . - . : -
. .
A
- 20 -
, .~ , . ..
. . ~. ~ . . ~ . .

AHP-6491
:
1~4~)625
- Also, the iinear rqduced form is obtained directly
by reduction of the cyclic undecapeptide of formula I t R =
NHRI and Rl is as defined above). Reduction with dithio-
threitol according to the method of W.W. eleland, Biochem.
3, 480 (1964) is preferred, although other agents known to be
effective for the reduction of cyclic disulfides to the
corresponding disulfhydryl derivative are appltcable, for example,
sodium blsulfite followed by hydrolysis of the intermediate
dJthtosulfate derivative.
tb) ComPounds I and la (R = H)
The requisite first peptide hydrazide viz., the tetra-
peptide of formula ~ Trt-S-CH CH(R)CO-Lys(80c)-Asn-Phe-Phe-NHNH2 ,
, in which R is hydrogen is prepared by reacting an activated ester
3 of 3-trltylth7opropionlc acid with a tetrapeptide of formula H- '! '
'
~ Lyst80c)-Asn-Phe-Phe-OMe to obtain the tetrapept7de of formula (2) 1` -
j In wh7ch R Is hydrogen, viz., Trt-SCH2CH2CO-Lys(Boc)-Asn-Phe-Phe-OMe
whlch Is subJect to hydrazlnolysis to obtaln said first tetrapeptide
hydraztde of formula t2) 7n wh7ch R is hydrogen.
In a preferred embodiment of the preparat70n of the above
Irst tetrapept7de~hydraz7de, the act7vated ester of 3-tr7tylth70-
prop70n7c ac7d, preferably the pentachlorophenyl ester, 7s preparsd
by combin7ng substantially equimolar amounts of 3-tritylthio-
prop70n7c ac7d, pentachlorophenol and d7cyclohexylcarbodiimide in
an tnert organTc solvent, preferably tetrahydrofuran , at about
0 to 10C. The mlxture is st7rred at about 0 to 10C for about
~' ~
one hour and then at about 20 to 30C for about one hour. The
m7xture 7s cooled to about 0C, f71tered and the f71trate
.
,. .
--21 -
i ~ . .- - . . -
.. ... .......

AHP-6491
1~40625
evaporated. The residue is crystallized to obtain 3-trityIthio-
propionic acid pentachlorophenyl ester. A solution of said last-
named compound and a substantially equimolar amount of the tetra- - ~ -
peptide of formula H-Lys(Boc)-Asn-Phe-Phe-OMe acetate, prepared
as described by H.U. Immer et al., cited above, in an inert
organlc solvent, preferably dimethylformamide or tetrahydrofuran,
ts treated with a substantially equimolar amount of an organic
base, preferably triethylamine, at about 20 to 30C. The mixture
ts sttrred for two to three days at about 20 to 30C and the
solvent evaporated under reduced pressure. The residue is -
triturated with cold dilute aqueous citric acid and water, dried,
and crysta-llized to give the tetrapeptide of formula Trt-SCH2CH2CO-
., :
Lys(80c)-Asn-Phe-Phe-OMe. SaTd last-named compound is dissolved
tn an inert organic solvent, for example, methanol, ethanol, or
."~
preferably dimethylformamide. The solutlon is treated with an
excess of hydrazlne hydrate, for example 15 to 30 molar equivalents.
.~ , .
~', The reaction mlxture is kept at about 20 to 30~C for about 20 to
~ 30 hours and evaporated under reduced pressure. The residue is ~;
'~ tr1turated with cold water and dried to give said tetrapeptide -
hydraztde of formula (2, R - H) Trt-SCH2CH2CO-Lys(Boc)-Asn-Phe-
Phe-NHNH2.
- 1 ~
In the next step of the process of this invention the
above first tetrapeptide hydrazide (2) and the second hexapeptide
of formula (3~ H-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-
NHCH CH S-Trt, described above in (a), are coupled accord7ng to the
~- 2 2
azide coupling method to obtain the corresponding linear deca- -~
. :~ - . -:peptide of formula (4) in which R 7s hydrogen, viz., the linear ~ -
:; : : '' ': ''
- 22
: -

AHP-6491
1~4~625
decapeptide Trt-SCH2CH2C0-Lys(Boc)-Asn-Phe-Phe-Trp-Lys~oc)-Thr(But)-
Ser(But)-NHCH2CH2S-Trt.
This coupling is conveniently achieved by dissolving the
first tetrapeptide hydrazide (2) in an organic solvent, preferably
a mixture of dimethyl sulfoxide and dimethylformamide, and cooling
the mixture to about -30 to -15-C. A solution of about two to
five molar equivalents of a strong acid, preferably three molar
equivalents of hydrogen chloride In ethyl acetate, is added to
the latter solution, followed by 1.0 to 1.5 molar equivalents of ;
an organTc nitrite, for example 1.2 molar equivalents of t-bùtyl -
nitrite. In this manner the corresponding tetrapeptide azide of
formula Trt-SCH2CH2C0-Lys(Boc)-Asn-Phe-Phe-N3 Ts obtained. After ,
about 15 minutes at about -20 to 0C a solution of substantially
one molar equivalent of the second hexapeptide (_) and an organic
base, preferably two to four molar equivalents of N-ethyldiiso-
propytam7ne, Tn an inert organic solvent, preferably dimethyl=
formamide, cooled to about -20 to 0C, is added to the above
solutlon contalning sald azide. The reaction mixture is then
stlrred at about -20 to 0C for one to two hours and then at
about 20 to 30C for about 20 to 30 hours and evaporated under
reduoed pressure. The residue is triturated with cold dilute
aqueous cttric acid, water, methanol and separation of the solid
gjves the aforementioned linear decapeptide (4, R = H).
The conversion of the above linear decapeptide to the
compound of formula I (R = H) is accomplished conveniently and
efficiently by flrst subjecting the last-mentioned linear
decapeptide to the action of iodine, preferably in the presence of
. .
,,
~' ' '~
~ - 23 -
~tl ~.... ' ' . . .. . . . . . .
'.~.~ . ' ' ` . ` ' i ' . :,

AHP-6491
;
.
~ )4~6Z5
methanol or acetic acid ~as described previously for the preparation
of the cyclic undecapeptide derivative in ta)], whereby re~oval of
-the sulfhydryl protecting group, i.e. Trt, and formation of the -
. .
disulfide bridge occurs to give the corresponding cyclic disul-
fide derivative of formula (5) in which R is hydrogen,
t t
Lys(~oc)-Asn-Phe-Phe-Trp-Lys(Boc~ThrtBu )-Phe-Thr(Bu )-
Ser(Bu )-NHC~2CH2 .
Subsequent treatment of the latter compound under
moderately acidic conditions, preferably concentrated hydrochloric
acid cooled to about 0C ~as described previously under (a)]
removes the remaining protecting groups (i.e., Boc and ~ut) to
,.. ~, , . :,
give the cyclic decapeptide of formula I (R = H) having the structure, j~
'', SCH2CH2CO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NHCH2CH2S
~. ,.
The linear reduced form of the latter cyclic decapeptide
Is obtatned preferentially by removal of the protecting groups from
the aforementloned llnear decapeptide of formula (4)in whlch R Is
h~drogen. Convenlent conditlons for thls deprotection step comprise
dissolvlng the linear decapeptide in concentrated hydrochloric acid
in the manner previously described under (a~. Alternatively, the !~
ITnear reduced form is obtained by direct reduction of the above
cyclic decapeptide of formula I (R = H) in the manner described ~ ;
under (a). . ¦-
'~ . ! '
~ Finally, it will be apparent to those skilled in the
.-~ , . .
art that. equlvalent amino, hydroxy or thiol protecting groups,
i~ equivalent methods of coupling peptide fragments, and equivalent ;
~3 ~ ~ methods for removal of amino, hydroxy or thiol protecting groups,
, other than those disclosed herein can be used in the embodiments
~ . - .
, ~ ~ , - ' ' ,
t
'`~' ,' ' ':
.'' ' , "
, .:
- 24 - I -

AHP-6491
1~4~6Z5
of this invention without departing from the scope and spirit
of the invention. Such apparent alternatives are intended to
be included within the scope of this invention.
The following flow diagram in which R is as defined in
the fTrst instance and Examples illustrate further this invention.
Trt-SCH2CHtR)CO-Lys(Boc)-Asn-Phe-Phe-NHNH2 (2)
; ~H~ s(8ec) Thr(Put)-Pho-Thr(8ut)-Ser(8ut)-NHCH~CH25-Trt (3)
'~ Linear peptide (4) Deprotection
,, ' '
. Oxidation \ I) Ag+ or Hg++ :
Z ¦ \ 2) H2S
....~Id~1on ~
Cyclic disulf1d,e derivatlve (5) Disulfhydryl derivative
Redu~tl~ I /
DeZpr~tectionDeprotectio ~ ~
'~ pept~de (I)Reduction > Linear reduced form of t
~ion peptide (la)
`~Z
1.
~, i
Z`: ' ' ' .
-
;! .
:
- 25 -
. .: . .. ~ .. . . .. .
. -. . :: . : . . .. : .

AHP-6491
1~4~6ZS
E~A~PLE I
Acetvl-(S-trityl) c ysteinvl-(N~-t-butoxYcarbonYl)lYsYI-as~araqinvl-
~henylalanyl-phenylalanine Methyl Ester . Ac-CYs(Trt)-Lys(Boc~-Asn- I
Phe-Phe-OMe
A solution of p-nitrophenyl acetate [0.191 9, 1.05 mmole,
prepared as described by F.D. Chattaway,-J. Chem. Soc., 2495
(1931)~ tn dimethylformamide (4 ml) is added to a solution at 0C
of H-Cys(Trt)-Lys(Boc)-Asn-Phe-Phe-OMe.HOAc~0.750 9, 0.698 mmole,
prepared as described by H.U. Immer et al., Helv. Chim. Acta., ¦ ~;
57, 730 (1974)] and N-ethylmorpholine tO.I ml). After stirring ¦
at 0C for 24 hr, the solvent is evaporated under reduced pressure. ~
The residue is dissolved in methanol (3 ml) and slowly added to ~ -
diethyl ether (200 ml). The precipitate is collected and ,;
~ crystallized from ethanol to give the tttle compound; mp
;, 219.5- 221C, ia~2D5 = -21.6 (c = 1, dimethylformamide).
-~ In the same manner by using the p-n7trophenyl esters of 1 -
formlc, proplonic, butyric, isobutyrlc, pivallc, n-hexanotc, or
~¦ ~ benzolc acid instead of p-nitrophenyl acetate, the corresponding ;~
compounds of the above formula in which Ac is replaced by formyl,
propionyl, n-butanoyl, isobutanoyl, pivaloyl, n-hexanoyl, or benzoyl
are also obtained. i~
.''i ~ ~ , : '` - .
:.
1'''~ . :. :'
.' . .
' " '~
- .
.; . .
,' ' ~' . . .'.':
-- 26 --

AHP-6491
1~4~1625
EXAMPLE 2
Acetyl-(S-tritvl) cvsteinvl-(N -t-butoxvcarbonYl)lvsYl-asParaqinY
phenylalanvl-phenylalanine Hydrazide ,Ac-Cvs(Trt)-LYs(Boc)-Asn-
Phe-Phe-NHNH2 (2, R = NHCOCH3)
A solution of Ac-Cys(Trt)-Lys(Boc)-Asn-Phe-Phe-OMe
(0.40 9, 0.379 mmole, described in Example 1) and hydrazine
hydrate (0.37 ml, 7.58 mmole) in methanol (15 ml) is stirred
at 0C for 48 hr. The precipitate is collected and dried to
give the title compound; mp 23~-237C, [a]2D5 = -28.6 (c = 1,
dimethylformamide).
In the same manner, by using the appropriate other
starttng materials described in Example 1, the corresponding
compounds of formula (2) in which R is NHRI wherein Rl is
formyl, propionyl, n-butanoyl, isobutanoyl, pivaloyl, n-hexanoyl,
or benzoyl are also obtained.
:, .
.1 6'
. .
~ ' ' , ' '
.`'' ' ~
.~ .
,, ~ ,
;~' '
~'J,
,`~ ' ' ,'
"
::
27
.
`
,. . . .. . .

AHP-6491
1~4~62~
EXAMPLE 3
Acetvl-tS-tritYI)cYsteinYl-(N~-t-butoxYcarbonYI)IysYI-asparaqinyl-phenyl-
alanYI-phenYlalanvl-trYpto~hYl tN6-t-butoxYcarbonyl)Iysyl-(O-t-butvl)
threonvl-phenylalanyl-(O-t-butvi)threonYI-(O-t-butyl)servl-2-tritylthio-'
ethylamide ~Ac-CYs!Trtj-L~s(Boc) Asn-Phe-Phe-Trp-LYs~Boc)-Thr(But)-Phe-
Thr(Bu )-Ser(Bu )-NHCH2__~5-Trt t4, R = NHCOCH3)
: .
A solution of Ac Cys(Trt)-Lys(Boc)-Asn-Phe-Phe-NHNH2 (2) (0.240 g,
0.227 mmole, descrTbed in Example 2) in dry dimethylformamTde (Z ml) and
dimethyl sulfoxide (lml) is cooled to -20C. Hydrochloric acid in ethyl
acetate ~2.1 N, 0.273 mmole) Ts added followed by t-butyl nitrite
(0.0312 ml, 0.273 mmole). The mixture Ts stTrred for 15 min at -15C.
A solution of H-Trp-Lys(Boc)-Thr(Bu )-Phe-Thr(Bu )-Ser(Bu )NHCH2CH2S-Trt(3,
O .304 9, 0.227 mmole, prepared as descrTbed in U.S. Patent
No. 3,917,581, cited above) in dimethylformamide (3 ml)- -
. .
contaTnTng N-ethyldTTsopropylamTne (0.097 ml, 0.568 mmole), cooled to
-15C, Is added dropwTse to the above reaction mixture. The mixture Ts
. ... .
stirred at -15C for one hr and at 25C for 20 hr. The solvent Ts
¦ evaporated under r~duced pressure. The resldue Is trlturated wlth ice
cold cltrTc acTd (I N) solutlon, flltered,washed wTth water and drTed
~ over phosphorous pentoxide. The solTd residue is trTturated wTth
i ~ ..
methanol, filtered and dried over phosphorous pentoxide to glve the
`, title compound, amino acTd analysis: ~ys, 1.82; Asp, 1.00; Ser, 0.76;
.~ , - , - -
1, Cyeteic acid, 0.93 Thr, 1.87; Phe, 3.10.
In the same manner, but uslng the appropriate other startlng
; materTals of formula (2) descrlbed in Example 2, the correspondTng
compounds of formula (4)Tn whTch R Ts NHRI wherein Rl is formyl,
propTonyl, n-butanoyl, Tsobutanoyl, pTvaloyl, n-hexanoyl, or benzoyl
are also obtaTned.
JI
~ . , ,
.::
' '
;~, , . . .
~ ~ - 28 -

AHP-6491
1~4~6ZS
EXAMP~E 4
CYCI jC disulfide of acetyl-cysteinyl-lysyl-as~araqinyl-phenylalanyl-
Dhenvlalanyl-tryptoDhyl-lvsyl-threonyl-~henylalanyl-threonyl-seryl-2
thloethylamide. Ac-Cjs-Lvs-Asn-Phe-Phe-TrP-LYs-Thr-Phe-Thr-Ser-NHCH ~ 2~
(1: R = NHCOCH ~ ;
A solution of Ac-Cys(Trt)-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr-
(Bu )-Phe-Thr(But)-Ser(But)-NHCH2CH2S-Trf (0.260 9, 0.110 mmole, described
In Examplé 3) in acetic acid (61 ml) is slowly added to a stirred solution
of iodine (0.278 g, 1.1 mmole) in methanol (56 ml) at 25C. After completion
of addition, the solution is stirred at 25C for one hr. The solution is
cooled to 0C and a solution of I N sodium thiosulfate in water is slowly
added to destroy the excess of iodine (colorless-solutjon). The solvent
is evaporated under reduced pressure and the residue triturated with water.
The precipitate is collected, washed with water and dried over phosphorous
pentoxide to give the cyclic protected undecapeptide of the formula
" . Ac-Cy ~ s(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(Bu )-SertBut)-
NHCH2CH2S (5, R = NHCOCH3).
1 The latter cyclic peptide is vigorously stirred at 0C under
an atmosphere of nitrogen for 10 min in concentrated hydrochloric acid
(9.1 ml1. Acetic acid (119 ml) is added and tne solution immediately
Iyophilized. The residue is dissolved in water and Iyophilized. The
resldue is dlssolved in the upper phase of the solvent system butanol-
acetic acid-water (4:1:5) and appl7ed to a column of a chemically modified
~'. cross-linked dextran (Sephadex G-25 M) prepared in the lower phase of the
solvent system. The upper phase of the above solvent system is used to
desorb the undecapaptide. The fractions containing the pure product àre -
combined and evaporated under reduced pressure. The residue is triturated
~ith diethyl ether, dissolved in 5% acetic acid and Iyophilized to give
the title compound as the acetic acid addition salt, U.V. (methanol):
max 29~ (6 4990), 282 (~ 5455), 274 nm (~ 5095).
, ` . ~:
., . ` ~ '
.
~ - 29 ` `
. .. . : . . ~ . . . .

- :
1~4~6Z5
The latter compound in the form of the acetic acid addition
salt is subjected to repeated Iyophilization from water to give the
title compound in the form of the free base, amino acid anaiysis: Lys, ~ ~
1.93; Asp, 1.00; Ser, 0.84; Cysteic acid, 0.80; Thr, 1.84; Phe, 2.97. :~ :
In the sa~e manner, by using the appropriate other start-
ing materials of formula (4) described in Example 3, the corresponding
compounds of formulae (5) and (1) in which R is NHRI wherein Rl is
formyl, propionyl, n-butanoyl, isobutanoyl, pivaloyl, n-hexanoyl or :
benzoyl are also obtained.
,
'', '',';
':i . - .
::
- . :
,
~ .
., :~ .
`~ ~ . ' - " , '
i: : ., '
: ; ' '
., I
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:"'. ,' .,' '
,, . ,:' '"
. - 30 -
.. , . . ~ , . - . . . . . ..... . .
.. . ... . . . . . . . . ..
,~ . . . . . . . ... . . . . .. .

AHP-6491
~.~4V625
EXAMPI;E 5
3-TritYIthioproDionic acid Pentach~orophenyl Ester (Trt-SCH~CH~COOPcP3
3-Tritylthiopropionic acid [1.0 9, 2.87 mmoles, described
by R.C.Hiskey and M.A. Harpold, J. Org. Chem., 33, 559 (1968)] is
dissolved in dry tetrahydrofuran t25 ml) and pentachlQrophenol tO.765 g,
2.87 mmoles~ is add~d. The mixture is cooled to 0C, dicyclohexyl-
carboditmide (0.596 g,2.87 mmoles) is added and the reaction is stirred
for I hr at 0C and I hr at 25C. The reaction mixture is then cooled
to 0C, flltered and the filtrate is evaporated under reduced pressure.
The residue is crystallized from ethyl acetate to give the title compound,
mp 154-156 C.
~ .
.~ .
~ . , , "' '
~ .
1 ~' . . .
,, ~ ~: , .
',1i~` . ':
:''.: ~ ~ , ' '
J, ~
:''` ' ~ ' ~'' '
'
_ 31 _
:: . ~ : , ~ . .
: ~, ~ ,. . .. . .
.; , , .

AHP-6491
1~14~6Z5
EXAMPLE 6
. .
3-Tritvlthio~roDionyl-(N~-t-buto);YcarbonYl)Iysvl-asparaqinvl-phenylalanyl-
phenylalanine MethYI Esteri Trt-SCH ~ _CO-LYs(Boc)-Asn-Phe-Phe-OMe
A solution of 3-tritylthiopropioniC acid pentachlorophenyl ester
.. . .
~- (0.597 9, I mmole, described in Example 5), H-Lys(Boc)-Asn-Phe-Phe-OMe-HOAc
[I mmole, prepared as descrîbed by H.U. Immer et al., Helv. Chim. Acta.,
~-~ .... . .
; 57, 730 (19741~ and triethylamine (O.14 ml, I mmole) is stirred at 25C -`
for 3 days. The solvent is evaporated under reduced pressure. The
residue is triturated with ice cold I N citric acid solution, fiItered,
washed with water and dried over potassium hydroxide. The solid is
crystaliiled from methanol to give the title compound, mp 215 - 220C.
., .
.:
, ,
.~ .
~ " . . .
. -, . . .
. :
.
. .
:.:, . .
~- $ ~
?: ::
g , ;
li i': : '
,.
'~ ',
:
i'i
- 32 -
~ , . . .. . .

AHP-6491
1~4~625
EXAMPLE 7
3-Tritvlthiopropionyl-(N -t-butoxycarbonyl)lvsyl-asparaqinyl-phenylalanyl-
phenylalanine HYdrazide Trt-SCH2 _2CO-LvstBoc)-Asn-Phe-Phe-NHNH2_(2, R = H)
A solution of Trt-SCH2CH2C0-Lys(80c)-Asn-Phe-Phe-OMe
(0.900 9, 0.9 mmole, described in Example 6j and hydrazine hydrate
(1 ml) in dimethylformamide (20 ml) is stirred at 25C for 20 hr.
The solvent is evaporated under reduced pressure. The residue is
trtturated with cold water, fiItered, washed with water and dried to
give the title compound, mp 225- 235C.
.,- . ' ~ ~ `
.,~ ~ ' '. ......... ~:
.''. .. . .
l,' , 1'`~ ~ "
. . . 1:.: :
. .,
.', . . - '
,',:
: 1:
,, ~ : '
- , '

AHP-6491
1~4~625
EXAMPLE 8
3-TritYlthiopropionyl-~N -t-butoxYcarbonvl)IysYl-asparaAqinyl-Dhenylalany
DhenylalanYI-trYptophYI-(N6-t-butoxYcarbonYl)lysvl-(0-t-butyl)threonYI-
phenYlalanyl-(O-t-butYI)threonv!-(O-t-butvl)serYI-2-trity ~hioeth ~am!de,
Trt-SCH ~ ~C0-Lys(Boc) Asn-Phe-Phe-Tr~-LYs(Boc)-Thr(Bu )-Phe-Thr(Bu )-Ser-
(Bu )-NHCH2__~S-Trt (4, R = H) -
A solution of Trt-SCH2CH2C0-Lys(Boc)-Asn-Phe-Phe-NHNH2 (0.500 9,
0.5 mmole, described in Example 7) in d7methyl sulfoxide (5 ml) and dimethyl-
formamlde (20 ml) is cooled to -20C. A solutton of hydrochloric actd In
ethyl acetate (1.4 N, 0.895 ml) is added followed by t-butyl nitrite (0.069 ml,
0.6 mmole). The mixture is stirred at -15C for 15 min and a solution, cooled ;
' to -15C, of H-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NHCH2CH25-Trt
(0.670 9, 0.5 mmole, prepared as described in U.S. Patent ~ ~
No. 3,917,581, cited above) and N-ethyldiisopropylamine - -
~ ~0.214 ml, 1.25 mmole) In dlmethylformamide (10 ml) is added. The
; reactlon mlxture is stlrred at -15C for I hr and at 25C for 20 hr and
i evaporated under reduced pressure. The resTdue is triturated with ice
cold I N cltric acld solu~lon, flltered, washed wlth water and dried
7 over phosphorous pentoxide. The solid Is triturated with cold methanol
and drled to give the tTtle compound, amino acid analys7s: Lys, 1.99;
Asp, I.IS; Thr, 1.73; Ser, 0.67; Phe, 3.00. ~ ~
~! -~ -- . . :
'{
''1, , '~' ~'' '' ' '
'; . . .. ..
i', '
~' ~ ~ ' '' ' ' ' ' .
` ~ ' ' :
34 -

AHP-6491
1~4~62~
EXAMPLE 9_
Cvclic disulfide of 3-thioDroDionYI-lYsYl-asDaraqinyl-phenylalanyl-Dhen
alanyl-trv~toPhY~ y-syl-threonyl---p-henylalan-~L-l-threonvl-se-ryl-2-thioethvl-H ~ ~C0-LYs-Asn-Phe-Phe-Trp-Lvs-Thr-Phe-Thr-Ser-N H ~H2~ -R = H)
.
A solution of Trt-SCH2CH2C0-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)
Thr(Bu )-Phe-Thr(Bu )-Ser(Bu )-NHCH2CH2S-Trt (0.500 9, 0.216 mmole,
described in Example 8) in acetic acid (100 ml) is slowly added to a
stirred solutlon of iodine (0.547 9, 2.16 mmoles) in methanol (110 ml) at
25C. After completion of addition, the solution is stirred at 25C for
, O .. .. ..
I hr. The solution is cooled to 0 C and a solution of l N sodi`um thiosulfate
in water is slowly added to destroy the excess iodine (colorless solution).
The solvent is evaporated under reduced pressure and the residue triturated
with water. The precipitate is collected, washed several times with
water and dried over phosphorous pentoxide to give the cyclic protected
decapeptide of formula SCH2CH2C0-Lys(Boc~-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-
Phe-Thr~But)-Ser(But)-NHC ~ S ~5, R = H).
The latter cyclic dec~peptide is vigorously stirred at 0C under
an atmosphere of ni~rogen for 10 min in concentrated hydrochloric acid
tl8 ml). Acetic acid ~200 ml) is added and the solution immediately
Iyophilized. The residue is dissolved in water and Iyophilized. The
.
residue is dissolved in the upper phase of tne solvent system butanol- -
. .
acetic acid-water (~ 5) and applied to a column of a chemically
modified cross-linked dextran (Sephadex G-25 M) prepared in the lower phase
, .... ,. . : . ..
of the solvont system. The upper phase of the above solvent system is
used to desorb the decapeptide. The fractions containing the pure
product are combined and evaporated under reduced pressure. The resTdue
is dissolved in 5% acetic acid and Iyophilized to give the title compound ;
as the acetic acid addition salt, U.V. (methanol): ~ max 290 tg 4920), 282
nm ( 6 5390).
. . .
.~.,, ~ . . : -.
'': ; ~
. . ~. .
~ - 35 - 1 ~
/ ~ i .

- AHP-6491
.
1e~4~625
, . , ii .
The latter compound in the form of the acetic acid addition salt .
is subjected to repeated Iyophilization from water to give the title compound
in the form of the free base, amino acid analysis: Lys, 1.. 97; Asp, 1.00; :
Thr, 1.64; Ser, 0.65; Phe, 2.94. .
', ~
i. ',
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